The invention relates to a process for the preparation of an antibiotic wherein a xcex2-lactam core is acylated and the antibiotic is recovered from the reaction mixture and subsequently the remaining mother liquor is worked up.
In the preparation of antibiotics involving the acylation of a xcex2-lactam core with an acylation agent, for instance a derivative of a D-phenyl glycine, the recovery of the antibiotic and the working up of the reaction mixture are difficult in general. Thus WO-A-93/12250 for instance describes that the acylation reaction never runs to completion and the ultimate purification of the final product is hindered because the acid/base properties and solubilities of some other components that are present (in particular 7-ADCA and phenyl glycine in the case of cephalexin preparation as described in U.S. Pat. No. 4,003,896) differ little from those of the final product. As a result, coprecipitation occurs, so that impure antibiotic, i.c. cephalexin, is obtained. In WO-A-93/12250 and U.S. Pat. No. 4,003,896 the use of a complexing agent such as naphthol is proposed. However, this entails the drawback that an additional substance alien to the process has to be added.
The objective of the invention is to provide a simple, general and widely applicable process, enabling the antibiotic to be recovered pure, without using such organic compounds that are foreign to the process and without large losses of costly (starting) material, i.c. xcex2-lactam core.
This is achieved according to the invention in that in the working-up step the mother liquorxe2x80x94which still contains a relatively high concentration of antibioticxe2x80x94is subjected to a hydrolysis reaction and subsequently the xcex2-lactam core is at least partially re-used for instance in an acylation reaction.
The acylation reaction can be effected either chemically or enzymatically. In the case of a chemical acylation and subsequent working up, racemisation will generally also result in the formation of a very small amount of acylation product with the wrong side chain enantiomer. If the acylation product in the mother liquor is recycled, strong accumulation of this side product may occur. The process according to the invention, with the acylation product and the acylation agent (side chain) being hydrolyzed again, offers the additional advantage of circumventing this problem.
The process according to the invention is preferably carried out in combination with an enzymatic acylation reaction. In the state-of-the-art processes a large excess of acylation agent has to be used in an enzymatic acylation reaction in order to ensure a high yield of antibiotic relative to xcex2-lactam core. This entails the drawback that either large losses of acylation agent occur as a result of hydrolysis or that a large quantity of the reaction mixture (that which remains after recovery of the antibiotic in the solid state) has to be worked up.
The applicant now has found a process wherein the use of a large excess of acylation agent during the acylation reaction is obviated and wherein nevertheless the xcex2-lactam core losses are limited, by subjecting the mother liquor to a hydrolysis reaction in which antibiotic still present in the mother liquor is decomposed into its initial compounds and the acylation agent is hydrolized as well, with the ability to subsequently recover or recycle the xcex2-lactam core. The fact is that it has appeared that the process according to the invention enables a simple process to be realized, with no need for full conversion of the xcex2-lactam core, while nevertheless only minor losses of xcex2-lactam core occur.
Surprisingly, it has been found that the solubility of the xcex2-lactam core is influenced by the presence of remaining acylation agent and antibiotic in the reaction mixture, in the sense that the solubility of the xcex2-lactam core is unexpectedly high at relatively high concentrations of acylation agent and antibiotic. As a result, it was found to be possible to recover virtually quantitatively the xcex2-lactam core in the solid state by hydrolizing the mother liquor obtained after isolation of the antibiotic, which results in hydrolysis of the antibiotic and the acylation agent. Moreover, it was found that the xcex2-lactam core, in the solid state, was recovered in a purer form than in a process in which hydrolysis of th)e mother liquor had not been effected. Especially the content of xcex1-substituted acetic acid in the xcex2-lactam core appeared to have been strongly decreased, which in turn had a surprisingly large effect on the acylation reaction speed. In particular, the content of phenylacetic acid in 7-ADCA and the content of phenyl or phenyloxy acetic acid in 6-APA appeared to be strongly decreased. In addition it appeared that the xcex2-lactam core crystals contained little adhering moisture, which means a little amount of impurities. An additional advantage appears in that, because of their low solubility, the xcex2-lactam core crystals could also be washed without large losses. This in turn, means a lower build-up of impurities, possibly accumulating in the antibiotic, in recycling.
The xcex2-lactam core obtained after the hydrolysis reaction constitutes a novel composition that has particular advantages in the enzymatic preparation of xcex2-lactam antibiotics. Applicant has found that the free phenyl or phenoxy acetic acid content in the xcex2-lactam core recovered after hydrolysis, is significantly lower than that of the xcex2-lactam core starting material, in particular it has appeared in relation to the 7-ADCA xcex2-lactam core that where the free phenylacetic acid content of the 7-ADCA starting material was 120 ppm, the free phenylacetic acid content of the 7-ADCA obtained after hydrolysis was 69 ppm; for 6-APA these figures were 30 ppm and  less than 15 ppm, respectively (ppm calculated with respect to the amount of xcex2-lactam core). The invention, therefore, also relates to 7-ADCA with less than 100, in particular less than 80, preferably less than 70 ppm phenylacetic acid, and to 6-APA with less 20, preferably less than 15 ppm xcex1-aryl- or aryloxy acetic acid, particularly xcex1-phenyl- or xcex1-phenyoxyacetic acid.
Another advantage of the process according to the invention is that as a rule thexe2x80x94generally valuablexe2x80x94hydrolized acylation agent can effectively be recovered.
Optionally, the xcex2-lactam core can be recycled in solution to the acylation reaction after the hydrolysis reaction. Preferably, the xcex2-lactam core is recovered, however, for instance by lowering the pH and isolating the xcex2-lactam core precipitated in the solid state. The hydrolized acylation agent can be recovered at various places in the process as a whole, for instance after the hydrolysis reaction, after the recovery of the xcex2-lactam core or after the condensation reaction.
The hydrolysis is preferably carried out in the presence of a suitable enzyme. Suitable enzymes for the enzymatic hydrolysis reaction are for instance the enzymes that are used in the preparation of xcex2-lactam cores and in the enzymatic acylation reactions, for instance amidases or acylases, in particular penicillin amidases or acylases. Such enzymes are described for instance by J. G. Shewale et al. in Process Biochemistry, August 1989, pp. 146-154, and in Process Biochemistry International, June 1990, pp. 97-103. Examples of suitable enzymes are enzymes derived from Acetobacter, in particular Acetobacter pasteurianum, Aeromones, Alcaligenes, in particular Alcaligenes faecalis, Aphanocladium, Bacillus sp., in particular Bacillus megaterium, Cephalosporium, Escherichia, in particular Escherichia coli, Flavobacterium, Kluyvera, Mycoplana, Protaminobacter, Pseudomonas en Xanthomonas, in particular Xanthomonas citril. 
Preferably an immobilized enzyme is used, since the enzyme can be easily isolated and re-used then. A suitable immobilization technology is described for instance in EP-A-222462. Another suitable technology consists in immobilizing the Penicillin G acylase on a carrier which contains a gelating agent, for instance gelatin, and a polymer with free amino groups, for instance alginate amine, chitosan or polyethylene imine. If besides the immobilized enzyme other solid substances are present as well, isolation can be effected with good results for instance by the method described in WO-A-9212782. Immobilized enzymes are known as such and are commercially available.
A suitable enzyme for the enzymatic hydrolysis reaction has appeared to be Penicillin-G acylase from Bacillus megaterium or Alcaligenes faecalis, described for instance in EP-A-453047. Also suitable are the Escherichia coli enzyme from Boehringer Mannheim GmbH, which is commercially available under the name xe2x80x98Enzygel(copyright)xe2x80x99, a Penicillin-G acylase from Escherichia coli, immobilized by means of the above-mentioned techniques, and the immobilized Penicillin-G acylase from Recordati.
A suitable method of recovering the antibiotic is for instance to lower the pH of the reaction mixture obtained after the acylation reaction, having a pH between 5 and 10, particularly between 6 and 10, preferably between 6 and 9, particularly between 7 and 8.5, thereby causing selective precipitation of the antibiotic; optionally, first the pH of the reaction mixture is brought to a value between 5 and 10, particularly between 6 and 10, preferably between 6 and 9 particularly between 7 and 8.5, and/or the solids are removed.
The optimum pH at which antibiotic is recovered depends on the composition of the mixture and is chosen such that optimum separation of xcex2-lactam core and antibiotic is achieved. In practice the optimum pH is a compromise between on the one hand high purity of the antibiotic recovered, which is achieved if the antibiotic is recovered at a relatively high pH, so that the antibiotic is still partly in solution and the xcex2-lactam core still completely in solution, and on the other hand a high yield, which is achieved if the pH at which the antibiotic is recovered is relatively low, so that the antibiotic has been precipitated virtually completely, while at the same time part of the xcex2-lactam core has also been precipitated. For the person skilled in the art it is easy to determine the optimum pH in a given situation.
The pH may be lowered in several ways in the framework of the invention, for instance this may be done chemically by adding an acid, for instance a mineral acid or a carboxylic acid, in particular sulphuric acid, hydrochloric acid, nitric acid, acetic acid or formic acid. Another possibility can be applied where for instance, if D-phenyl glycine amide (PGA) has been used as acylation agent in the acylation reaction or if an ester of D-phenyl glycine (PGM) has been used and the pH has been kept constant by means of titration with ammonia during the acylation reaction. In such case the pH can be lowered through physical removal of ammonia. Suitable physical removal methods include for instance stripping with steam or an inert gas; (steam) distillation at reduced pressure, in particular thin-film evaporation; evaporation in a spray tower; gas membrane separation or electrodialysis.
The temperature at which the hydrolysis reaction is carried out is not particularly critical and is preferably between 0 and 50xc2x0 C., in particular between 5 and 40xc2x0 C., preferably between 15 and 30xc2x0 C.
The pH at which the hydrolysis reaction is carried out is not particularly critical and is preferably between 6 and 9, in particular between 7 and 8.
The process according to the invention can be suitably applied for the preparation of xcex2-lactam antibiotics, for instance cefalexin, amoxicillin, ampicillin, cefaclor, cefradin, cefadroxil and cefotaxim, cefazolin and the like.
Suitable examples of xcex2-lactam cores which can be used according to the invention are various penicillanic acid derivatives, for instance 6-aminopenicillanic acid (6-APA), and cephalosporanic acid derivatives, for instance 7-aminocephalosporanic acid, with or without a substituent at the 3-site (7-ACA), for instance 7-aminodesacetoxycephalosporanic acid (7-ADCA) and 7-amino-3-chlorocephalosporanic acid (7-ACCA).
In the (enzymatic) acylation reaction, the acylation agent can be for instance a phenyl glycine in activated form, preferably an amide or an ester, for instance a methyl ester; suitable phenyl glycines are for instance substituted or non-substituted phenyl glycines, in particular phenyl glycine, p-hydroxyphenyl glycine, dihydrophenyl glycine.
The enzymatic acylation reaction is mostly carried out at a temperature lower than 40xc2x0 C., preferably between 0 and 35xc2x0 C. The pH at which the enzymatic acylation reaction is carried out is mostly between 5 and 10, particularly between 6 and 10, preferably between 6 and 9, particularly between 6.5 and 9.
In practice the (enzymatic) acylation reaction and the further working up of the reaction mixture are mostly carried out in water. Optionally, the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30 vol. %. Examples of organic solvents that can be used are alcohols with 1-7 carbon atoms, for instance a monoalcohol, in particular methanol or ethanol; a diol, in particular ethylene glycol or a triol, in particular glycerol.
An embodiment of the process according to the invention is worked out in detail in the following, relating to the preparation of cephalexin from 7-ADCA and D-phenyl glycine amide (PGA). In this embodiment a reaction mixture obtained after an enzymatic acylation reaction at a pH between 8 and 10 and containing cephalexin, 7-ADCA, PGA and D-phenyl glycine (PG), is treated to isolate the solid it contains, which solid mainly consists of the immobilized enzyme and possibly PG. The pH of the remaining liquid mixture is then lowered to 6-8, particularly to 6.5-7.5, resulting in the formation of a precipitate which mainly contains cephalexin, after which the solid cephalexin is recovered. In this preferred embodiment the then remaining liquid mixture is subjected to an enzymatic hydrolysis, resulting in hydrolysis in particular of the remaining, dissolved cephalexin and the remaining PGA to 7-ADCA and PG and to PG, respectively; the PG precipitates virtually completely and can be isolated, for instance by filtration. It so desired the PG can be removed (in whole or partly) already during the hydrolysis reaction by circulation of the supsension over a filter outside the reactor. Next, the 7-ADCA can be separated from the then remaining mixture for instance after a further lowering of the pH, thereby causing the 7-ADCA to precipitate; it can then be isolated, for instance by filtration or centrifugation, and used again in an acylation reaction.
In the framework of the present invention the various components may be present in the reaction mixture in the free form or as salts. The pH values mentioned are in all cases the pH values measured with a pH electrode calibrated at room temperature.
The invention will be further elucidated by means of the following examples, without however being restricted thereto.
Abbreviations